Arginine is a conditionally essential amino acid that has many functional roles besides protein synthesis. Arginine is utilized for the synthesis of creatine, polyamines and nitric oxide and thus has a central role in energy metabolism, cell proliferation and in the regulation of blood pressure and immune response. Arginine supplementation has been beneficial in a variety of circumstances such as preeclampsia, hypertension, and sepsis. Detrimental outcomes, however, have been reported, and the long term safety and efficacy of arginine therapy remain poorly described. Citrulline, the precursor for endogenous arginine synthesis, is a viable alternative to arginine supplementation. Whereas first pass extraction and hydrolysis of arginine is considerable (~40%, and likely to increase due to supplementation) citrulline escapes liver metabolism. For this reason citrulline supplementation has the potential to deliver more arginine to peripheral tissues than arginine supplementation itself. Moreover, different cell types have the ability to directly utilize citrullne to meet their local arginine needs. This ability of cells to directly utilize citrulline is the reasonwhy cancer patients treated with pegylated arginine deiminase (ADI-PEG), which results in arginine depletion but high levels of citrulline, are able to maintain metabolic homeostasis. Citrulline supplementation has the added advantage that it can provide arginine when arginine availability is compromised due to high arginase activity in the gut and circulation (e.g., inflammatory bowel disease, sickle cell anemia hemolysis). To address the hypothesis that supplemental citrulline is a better source of endogenous arginine availability than arginine supplementation itself, we propose the following specific aims. Specific Aim 1: To determine the pharmacokinetics, oxidation and the first pass metabolism of arginine when arginine or citrulline are chronically supplemented in mice. Specific Aim 2: To determine the molecular basis for increased citrulline utilization when arginine is limiting and to determine fractional protein synthesis rate of differet tissues, whole body nitric oxide production and blood pressure in ADI-PEG arginine-depleted mice. Specific Aim 3: To determine the reduction in blood pressure in a chronic hypertensive mouse model and in a mouse model of preeclampsia supplemented with different levels of arginine and citrulline. Different metabolic (fluxes, oxidation, metabolite interconversion) and physiological (blood pressure, NO synthesis, protein synthesis) endpoints will be measured utilizing stable isotopes and blood pressure telemetry. Our current knowledge can only account for ~60% of the citrulline produced as the precursor for plasma arginine. Because the only known fate of citrulline is its conversion into arginine the unaccounted citrulline is likely to provide the precursor for local arginine synthesis. These studies will fill the gap in our knowledge regarding this unaccounted citrulline and its physiological effect. Moreover this information will provide the basis for new human therapeutic trials and recommendations on arginine and citrulline supplementation when the demand for arginine is increased as in hypertension, preeclampsia, sepsis, inflammatory bowel disease, and sickle cell anemia.